Aml. Chem. 1991, 63, 1971-1978
Figures 6 and 7. The extent to which these random target compounds exist in pyrolysis products from an arson scene establishes a limit below which accelerant patterns would become unrecognizable.
CONCLUSIONS Target compound analysis is a useful approach to the identification of residual petroleum products in fire debris. The generation of target compound chromatograms facilitates this process. Target compound patterns for fresh and weathered gasoline, MPD, and HPD are sufficiently specific to allow their identificationin high-background arson samples. Registry No. 1,3,5-Trimethylbenzene, 108-67-8; 1,2,4-trimethylbenzene,95-63-6; 1,2,3-trimethylbenzene, 52673-8; indane, 496-11-7; 1,2,4,5-tetramethylbenzene,95-93-2; 1,2,3,5-tetramethylbenzene, 527-53-7; 5-methylindane, 874-35-1; 4-methylindane, 824-22-6;dodecane, 112-40-3; 4,7-dimethylindane,668271-9; 2-methyhphthalene, 91-57-6,l-methylnaphthalene,90-12-0; ethylnaphthalene, 27138-19-8; l,%dimethylnaphthalene,575-41-7; 2,3-dimethylnaphthalene,581-40-8; decane, 124-18-5; n-butylcyclohexane, 1678-93-9; trans-decalin, 493-02-7; undecane, 1120-21-4;n-pentylcyclohexane,4292-92-6; n-hexylcyclohexane, 4292-75-5;tridecane, 629-50-5; n-heptylcyclohexane, 5617-41-4; tetradecane, 629-59-4; n-octylcyclohexane, 1795-15-9;2,3,5-trimethylnaphthalene, 2245-38-7; pentadecane, 629-62-9; n-nonylcyclohexane, 2883-02-5; hexadecane, 544-76-3; heptadecane, 629-78-7; pristane, 1921-70-6; octadecane, 593-45-3; phytane, 638-36-8; nonadecane, 629-92-5; eicosane, 112-95-8; heneicosane, 629-94-7.
1971
LITERATURE CITED D. M. J . ~ o n m s l ~ IBW, ~ d . 5,236-247. Parker, B. P.; Rajeswaran, P.; Kkk, P. L. M”. J . 1862, 8 , 31-36. Mldklff, C. R.; Washington, W. D. J . Assoc. Otf. Anal. CY”. 1072, 55, 840-845. Yip, I . H. L.; Clalr, E. G. a n . Soc. Fawrplc Sd. J . 1078, S,75-80. L-S,
Bertsch,W.;Sdbrs,C.S.J.Hk3,R~.CYwomefog*.Clvomefog*. Commun. 1888, 9 , 657-661. DeHaan. J. D.; Bonarlus, K. J . F m s b Sd. Soc. 1888, 28, 299-309. Smith, R. M. Anel. Chem. 1082, 54, 1399A-1409A. Juhela. J. A. A m Anel. New. 1070, 3 , 1-19, Hober, 0.; Bertsch, W. Am. Lab. 1088, 20, 15-19. Aldrldge, T. A.; OeteS, M. J . Fwenslc Sd. 1886, 31, 666-686. Wlneman, P. L. (unpublished work) Symposium on Recent Advanes In Arson Analysh and Detection, Lee Vegas, NV, 1985. Bertsch, W.; Sellers, C. S.; Babln, K.; Mer, G. J . M& RSSOM. chroma-. chsomew. COmmM. 1888, 1 1 , 815-819. Kelly, R. L.; Mae, R. M. J . FcmMslc Sd. 1084, 20, 714-722. HWes, R. A.; Blemann, K. Anel. Chem. 1070, 42, 855-860. Fwenslc Sclence and Englneerkrg Commmee of the Intematknel Association of Arson Inwstlgators. Rg Areon Invesf@rOr 1888, 98 (4), 45-48. Chrostowski, J. E.; Holmes. R. N. Arson Anel. New/. 1878, 3 (5), 1-17. Tontarskl, R. E.; Strobel, R. S. J . FonmslC Sd. 1082. 27, 710-714. &ob, K. CIesSicel Sph end Spnmness In)sction In &pI&ry Q.C, 2nd ed.;Huthlg: New Yak, 1968; pp 56-58, 97. Sanders, W. N.; Maynard, J. 8. Anal. 0”.1868, 40, 527-535. Irwin, W. J. Anel)arcelpyrdysls; Marcel Dekker: New York, 1982; pp 133- 134. Hunt, J. M. P e m &oChedsby end oedogy;W. H. Freeman and CO.: San Franclsco, CA, 1979; pp 89, 101.
RECEIVED for review January 17,1991. Accepted June 7,1991.
Charge Determination of Product Ions Formed from Collision- Induced Dissociation of Multiply Protonated Molecules via Ion/Molecule Reactions Scott A. McLuckey,* Gary L. Glish, and Gary J. Van Berkel Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365
of bn/mokcule reactknr lnvolvlng muttiply protomid bnsdorlved from dectmpray for the detennhakn of the c h a r m of product bns fonned from colkkn-lnducd d b dation b The expwlmenb are carded out wiih a quadrupole Ion trap capable of muttlple siages of mass spectrometry. The approach k W a i d wlth proton transfer from a product Ion from quadruply proionaied meliiiln, and 20H)* lon from horse from a product b n from ihe (M myOgMlh, io l,edhhohex8nO. The major product ion hwn quadruply proionaied bovine lnsulln Is used io lllusiraie the use of a clustering reaw h 1,tkllamlnOhexane. The Ion trap Is shown io be a parilcularly useful tool for employing both collkknal acthraibn and bw-energy lon/mdecule reactknr hthe”eexp.rhmtiodet.mrlnproduct ioncharge. T)n w#)
+
INTRODUCTION Fenn and co-workers fiit demonstrated the propensity for multiple cationization of poly(ethy1ene glycols) (1) and biopolymers, such as peptides and proteins (2,3),via electrospray (ES).Since then, numerous examples of the usefulness of ES as a source of multiply charged ions for mass spectrometry 0003-2700/9 1/0363-197 1$02.50/0
have appeared. Much of this work has recently been reviewed (4-8). Biopolymers, in particular, show a strong tendency for multiple protonation (e.g., proteins) or deprotonation (e.g., oligonucleotides),resulting in highly charged ions. Although the ability to form highly charged ions provides new opportunities for mam spectzometry, the analysis of these ions poeee new challenges. Among these is the establishment of ion charge. Most maas spectrometers provide the ion mass/charge ratio or some property proportional to mass/charge, such as momentum/charge or kinetic energy/charge. The majority of ionization methods provide predominantly singly charged ions, which makes trivial the determination of mass. The number of charges aasociated with ions formed via ES,on the other hand, is highly variable and can range up to many dozens of charges. Fortunately, a distribution of charge states is typically observed with ES for molecules that tend to form multiply charged ions. Furthermore, little or no fragmentation is typically observed for ions derived from ES. As a result, the ES masa spectrum of a multiply protonated polymer shows a series of peaks, each of which differs from its adjacent peaka by 1unit charge, and by 1Da. The procedure for obtaining the molecular weight of the polymer from the ES mass spectrum is straightforward and has been described (3,9,10). 0 I991 Amerlcan Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991
The ES mass spectrum gives molecular weight information but, due to the lack of fragmentation, provides no structural information. A now standard approach to obtaining structural information from polyatomic ions is to subject them to mass spectrometry/mass spectrometry (MS/MS) (11,22). To date, the majority of reports involving MS/MS analysis of ions derived from ES have been performed with triple quadrupole mass spectrometers (6,13-18). These include the pioneering studies of the collision-induced dissociation (CID) of highmass, multiply charged biopolymers by Smith et al. (6,15-18). One of the many important observations of Smith's group is the difficulty often encountered in interpreting MS/MS spectra of multiply protonated parent ions due to uncertainty in product ion charge. When two, and only two, product ions are formed upon CID of a single parent ion and both appear in the MS/MS spectrum, charge states can be assigned from the known charge of the parent ion and differences in mass/charge of product ions from that of the parent ion. However, both complementary ions, that is, both ions expeded from a single dissociation, are often not observed in ESMS/MS spectra of multiply charged ions. This has proved to be a common observation for peptides and proteins, for example (6, 15-18). An approach to the determination of ion charge is to measure the mass/charge positions of the isotope peaks. This approach has been demonstrated for ES mass spectra of peptides and proteins, for example, by Henry and McLafferty (19) with a Fourier transform mass spectrometer and for ESMS and ESMS/MS spectra by Schwartz et al. (20) by using a quadrupole ion trap. Mass resolution in excess of M/AM = 30000 at m / z 502 has been demonstrated in the quadrupole ion trap by reducing the mass scan rate (20). This constitutes an improvement of slightly more than an order of magnitude in the mass resolving power of the commercially available quadrupole ion trap. The compromises necessary to achieve such resolving power in practical applications, however, have yet to be fully characterized or addressed. Furthermore, cases may arise where resolving power is not suffciently high or may not permit charge state assignment either due to sensitivity considerations or to isobaric product ions. Some other approach to product ion charge state determination with the quadrupole ion trap might therefore be useful in order to more fully take advantage of its capabilities as an MSf MS and MS" instrument. A possible approach to product ion charge state determination that may complement, or substitute for, high-mass resolution is to alter the mass or charge of the ion in a known fashion. This might be done in an MS3 experiment whereby the parent ion is subjected to CID and the product ion is allowed to undergo an ion/molecule reaction. For multiply protonated ions, a proton-transfer reaction to a neutral base would result in a unit change in charge and a concomitant change in mass of 1 Da. Alternatively, a clustering reaction in which a base forms a proton-bound dimer with the product ion would result in a change in mass equal to the mass of the base. Either approach would allow the charge state to be determined. ES has recently been coupled with the quadrupole ion trap, and MS/MS and MS" experiments involving CID with multiply charged parent ions have been reported (21-23). We have recently reported MS/MS experiments involving proton-transfer reactions of multiply protonated proteins with a neutral gas-phase base (24) and noted also the observation of clustering with some of the ions. This paper describes the use of ion/molecule reactions for charge state determination of product ions formed by CID of multiply protonated molecules in a quadrupole ion trap. EXPERIMENTAL SECTION All experiments were carried out with an ion trap mass spec-
trometer (M) manufactured by Finnigan (San JOW,CA), which has been modified to allow ions to be injected from an external ion source (25).The hardware for an atmospheric sampling glow discharge ion source (26) is used as the ES interface. The modifications and operating procedures used to sample ions from atmosphere and to inject them into the ion trap have been described (21,27). All experiments involving ion/molde reactions were performed in the presence of l,&diaminohexane (MW 116) admitted into the vacuum system via a leak valve to estimated pressures of (1-3) x IO-' Torr. The pressure was adjusted to provide number densities of the base sufficiently high so that ion/molecule reactions would be 50-100% complete within 0.1-0.3 5.
All ion trap experiments consist of a sequence of steps beginning with an ion injection period and ending with a scan in which ions are mass selectively ejected from the ion trap and into an electron multiplier detector. The overall experimental sequence is often referred to as the scan function, which includes the order, duration, and magnitudes of the signals applied to the ion trap electrodes, as well as coordinated events, such as ion injection. The operation of the ion trap in analyzing multiply charged ions has been described in detail (21) including the procedure for CID and mass analysis. The method used to isolate ions with m / z values beyond the normal mass range of the ion trap has also been described (28). Note that all experiments described herein use yresonance ejection" of the ions in mass analysis (29,30)with the standard ring electrode rf amplitude scan rate provided by the ITMS electronics. The effective mass scan rate for these experiments was 22 222 Da/s. As described elsewhere (211,mass accuracy and mass resolution suffer at these scan speeds. Means to allow for variable scanning speeds at any mass range have not been incorporated into the system for routine use. A unique 8 c ~ function n was used for each of the experiments described herein, and these are briefly described below according to the molecule to which they apply. Scan Functions for Melittin Experiments. All experiments included an ion injection period of 200 ms and an ion isolation step to isolate the (M + 4 H)'+ ion ( m / z 848.5). For MS/MS experiments involving proton-transfer reactions, a variable time delay after ion isolation and before the mass scan was used to allow reactions to occur. For MSa experiments, a supplementary rf signal of the appropriate frequency and amplitude (typically 1 V p-p) was applied for 20 ms immediately after the isolation of (M + 4H)'+ to effect its CID. A second ion isolation step was then used to isolate the product ion of interest followed by a variable delay period to allow ion/molecule reactions to proceed. The mass scan completes the experimental sequence. The total time for a single execution of the scan function was 280-430 me depending upon the length of the ion/molecule reaction period. Each of the displayed spectra are the average of 30 scans. Scan Functions for Bovine Insulin Experiments. Mass spectra were acquired by using an ion injection period of 200 me followed by a delay period of either 10 ms or 300 ms before the mass scan depending upon whether or not ion/molecule reactions were of interest. All MS/MS and MS3 experiments were performed by using an ion injection period of 200 ms followed by an ion isolation step to isolate as parent ions either (M + 5 H)w ( m / z 1147.8) or (M + 4 H)'+ ( m / z 1434.5). The MS/MS experiments involving ion/molecule reactions employed a delay of 200 ms prior to the final mass scan. For the MSs experiments, isolated parent ions were subjected to CID followed by isolation of the product ion of interest and a variable reaction period up to 100 ms prior to the final mass analysis step. The total time for a single execution of an MSs experiment was 280-380 ms depending upon the length of the ion/molecule reaction period. Spectra are the average of 10-20 scans. Scan Functions for Myoglobin Experiments. All myoglobin experiments were performed by using an ion injection period of 350 ms. This period was followed by an 8-msramp of the ring electrode rf amplitude from 577 to 7500 V 0-p while applying a 6-V p-p rf signal of 90.5 kHz to the end caps to promote desolvation of the injected ions. The present ES source and interfaca employ no measures to intentionally dry ions prior to introducing them into the ion trap. Although significant drying can occur within the ion trap (21),we have noted that proteins sometimes do not completely desolvate on the time scale of the typical ion
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991
1978
isolate (M+4H)4+
g031
4
ion injection
lbo
j
react
200
360
scan
1
460
Time (msec) 750
'
1050
'
1350
'
mlz 9031
m
mlz
Figure 2.
(a)Variation of the ring electrode rf amplitude as a function
of time lndicatlng the ste s of an MSlMS experiment whereby the mass-selected (M 4 H)fi ion or melittin was atibwed to react with 1,6diamhohexane. (b) W/MS specbum resulthg from the experiment indicated in (a) using a 20bn/rolecule reaction period. The b w mass portion of the spectrum (not shown) indicates a concomitant increase in protonated l,&diamInohexane with (M 3 H)".
+
+
mh Figure 1. ES mass spectra of horse myoglobin acquired (a) without and (b) with a desolvation step prior to mass analysis.
trap experiment. The step in the scan functino just described brings all ions sequentially into resonance with the signal applied to the end caps, thereby kinetically exciting them. The ramp is sufficiently short to prevent ion ejection but sufficiently long to increase the energy of collisions between the ions and the background helium, thereby assisting in desolvation. The effect of this "heating" step is shown in Figure 1. Figure l a shows the mass spectrum of horse skeletal muscle myoglobin acquired without the heating ramp, and Figure l b shows the maas spectrum acquired under identical conditions but with the heating ramp. The broad, poorly resolved trace in Figure la reflects extensive solvation in which ion signals are spread more or less uniformly over the indicated m / z range. Mass spectra were acquired by scanning the ion trap directly after the heating ramp. For MS/MS spectra involving CID, the heating ramp was followed by isolation and collisional activation of the (M+ 20 H)locparent ions prior to the final mass scan. For MS3 experiments, the m / z 884 product ion formed during the collisional activation step was isolated and allowed to react for up to 200 ma with the base prior to the final maas analysis step. The total time for a single acquisition was 680-880 ms depending upon the length of the ion/molecule reaction period. Spectra are the average of 20-40 scans. Materials. Melittin, bovine insulin, and horse skeletal muscle myoglobin were obtained commerciallyand were used as received to prepare solutions for electrospray. Each compound was dissolved in a mixture of HPLC-grade methanol, water, and glacial acetic acid in relative proportions of 75%, 20%, and 5% by volume, respectively, to give a concentration of (2-8) X lo4 M. All solutions were infused through a dome-tipped 120-pm4.d. stainleas steel needle at a rate of 0.5-1.0 pL/min while the needle was held at a potential of +3.+3.5 kV. A few crystals of 1,6diaminohexane were placed in a vial connected to the vacuum system via a variable leak valve. The room-temperature vapor pressure of this base is sufficiently high to give the requisite pressure in the vacuum system via adjustment of the leak valve. RESULTS AND DISCUSSION The results described here were selected to illustrate the phenomenology and practical use of reactions of multiply protonated ions with a gas-phase base. Reaction rates, rate constants, intermediate lifetimes, etc., for these reactions will
be described in detail elsewhere. One of three eventualities, viz., clustering between the ion and one or more molecules of the base, proton transfer to the base, or no reaction, is observed from the low-energy ion/ base collisions that occur within the ion trap. In each case, a proton-bound complex between the ion and the base constitutes a local minimum along the reaction coordinate. If the activation energy to products is significantly higher than the reactant energy (potential and kinetic), the proton-bound commplex is only weakly bound and cannot survive the 400-700 K effective ion temperatures that exist within the ion trap (31).In this case, no reaction is observed. If the activation energy to products is below the energy of the reactants by several kilocalories/ mole, the lifetime of the proton-bound complex is short relative to collisional relaxation by helium and proton transfer is observed. If the activation energy to products and the energy of the reactants are nearly equal, the lifetime of the proton-bound complex is maximized and collisional relaxation of the complex becomes significant, resulting in the observation of clustering. The outcome of the interaction is largely dependent upon the strengths of the binding sites on the ion, the strength of the base, and the Coulombic field in the multiply charged system. To maximize the possibility for a reaction, it is desirable to use a strong gas-phase base. It is for this reason that 1,8diaminohexane was chosen for these studies since it is a strong gas-phase base (proton affinnity = 235 kcal/mol (32))that is conveniently admitted into the vacuum system. Melittin. The ES mass spectrum of melittin shows predominantly the (M + 4 H)'+ ion with intensities of the (M + 5 H)s+ and (M + 3 H)3+ ions consistently less than 20% that of the base peak. Figure 2 shows the MS/MS spectrum obtained when the (M + 4 H)'+ ion is mass-selected and allowed to react with the base for 200 ma. The figure also shows the variation of the ring electrode rf amplitude with time, indicating the various steps in the experiment. Proton transfer is observed with virtually all of the loss in (M + 4 HI4+signal accounted for in the (M + 3 HI3+signal. On the other hand, a similar experiment in which the (M + 3 H)3+ ion is isolated for reaction showed neither proton transfer nor clustering. The ion trap MS/MS spectrum of the (M + 4 H)'+ ion involving CID has already been reported (21). It shows a
1974
ANALYTICAL CHEMISTRY, VOL. 63,NO. 18, SEPTEMBER 15, 1991
Chart I
B ~ I l a j l G l yA - l a - Y I I - L e u ~ " a l - ~ e u - T h r - G l y -Leu
rY8
Flgure 3. MSsspectra resulting from mass selection and CID of the (M 4 H)'+ bn of mellttin followed by mass selectbn of the product bn at m l z 542 and an ionlmolecule reaction period of (a) 20 ms and (b) 150 ms.
+
variety of predominantly B and Y type ions with charges of 1-3. The ion trap spectrum is remarkably similar to that reported in a triple quadrupole study (33)in which all of the possible A, B, C, X, Y, Z products (34)were listed. Charge state assignments were made through the laborious process of matching the observed product ion m / z ratios with the list of all of the possible products from the known sequence of the peptide. Figure 3 illustrates charge state assignment via an ion/molecule reaction as part of an MS3 experiment. The product ion from CID of (M + 4 H)4+ at m/z 542 was mass-selected and subjected to reaction with the base over various reaction periods. A 20-ms reaction time experiment (Figure 3a) shows the MS3 spectrum under conditions where an ion/molecule reaction cannot proceed to a significant extent. A 150-ms reaction time experiment (Figure 3b) shows the product ion at m/z 542 shifta almost quantitatively to the second generation product ion at m/z 812. With these two m/z values and the assumption that they differ in unit charge by 1and in mass by 1Da, the charge states can be assigned as has been described (IO). This procedure indicates that the charges are +3 and +2, respectively, and support their assignments,based on the known structure of melittin, as Y,33+ and Y13'+. The preceding example illustrates that proton-transfer reactions can provide charge state information without a significant loss in sensitivity. The efficiency of the ion/ molecule reaction was nearly 100% and the duty cycle (i.e., the ratio of the ion injection time to the total time for execution of the scan function) was reduced by less than a factor of 2. Furthermore, the reaction need not be run to completion to obtain the desired information so that shorter reaction times can be used. However, the Y1a3+ion is the only product ion from CID of the (M + 4 HI4+parent that reacts rapidly with 1,6-diaminohexaneunder these conditions. Most of the other product ions are believed to be singly or doubly charged. The only other product ion observed in the ion trap CID experiment believed to be triply charged is the Yu3+ ion, and this ion is unreactive. This illustrates that, despite its relatively high basicity, 1,6-diaminohexaneis not universally reactive with multiply charged ions. A stronger gas-phase base is necessary to expand the applicability of proton-transfer reactions for charge state determination. Aside from the determination of charge states, the relative reactivities of the ions derived from melittin may provide information on the sites of protonation. The reasonable assumption is often made when the number of charges is less than or equal to the number of the most basic sites, viz., the
C D
E
1
F
side chains of basic residues lysine, histidine, and arginine and the N-terminus, that the protons me distributed on theae sites. Chart I shows the amino acid sequence of melittin with the most likely protonation sites indicated as A-F. The chart also indicates the sequences of the Yu,Y13,and Ye products. Upon formation of a Y product, a new N-terminus is formed at the site of fragmentation. For the quadruply protonated molecule, no combination of protonated sites can avoid two protons in the Lys-Arg-Lye-Arg portion of the sequence. The triply charged molecule, on the other hand, can be protonated at the N-terminus, the lone Lys, and in the Lys-Arg-Lys-Arg sequence, which are relatively remote from one another. The rapid proton transfer between (M+ 4 H)'+ and the base and the lack of reactivity of (M+ 3 HI3+ may be due to strong Coulombic repulsion destabilizing the protons in the LysArg-Lys-Arg sequence in the former. The behavior of the triply charged products from (M+ 4 H)'+ is consistent with this speculation. No combination of charged sites in the Y 1:+ product can avoid at least two protons in the Lys-Arg-Lys-Arg sequence, whereas triply charged Yu can be protonated at the N-terminus, the lone Lys, and somewhere within the LysArg-Lys-Arg sequence. The former reacts rapidly whereas the latter does not. This particular observation shows that the number of charges alone does not determine reactivity but also that their relative locations are important, as expected. The relatively unfavored condition of two protons in the Lys-Arg-Lys-Argsequence is also reflected by the absence of any doubly charged Y, ions, where n < 8, in the CID MS/MS spectrum of the (M 4 H)" parent. For the larger Y products, at least five amino acid residues separate the Nterminus from the Arg nearest the C-terminus. All of these observations support the expectation that the most thermodynamically stable multiply charged ions tend to have the protons spaced to minimize repulsion. It should be emphasized here that the lifetimes of the ions sampled in the ion trap experiment are on the order of hundreds of milliseconds. It is therefore expected that intramolecular proton transfer yields predominantly the most stable proton distributions, at least for smaller peptides and proteins. Bovine Insulin. Figure 4 compares ES mass spectra of bovine insulin acquired in the absence of the base and in the presence of the base with an additional 0.3 s delay after ion injection to allow ion/molecule reactions to proceed. After the reaction period, the (M+ 5 HI6+ion is seen to be largely displaced to quadruply charged ions and the protonated base signal (m/z 117) increases concurrently. a minor amount of clustering is also observed for (M + 5 HIM. A relatively small degree of proton transfer from the quadruply protonated ion to the triply charged ion is observed. In contrast to the (M + 5 H)s+ion, the major reaction for the quadruply protonatd ion is clustering with the base, fiist to form the (M + 4 H + B)'+ ion and then to add more molecules of the base. Figure 5b, which shows a portion of the MS/MS spectrum acquired after the (M + 4 H)'+ ion was mass-selected and allowed to react with 1,6-diaminohexanefor 0.3 s, illustrates this behavior. The spectrum shows a series of peaks comprised of the unsolvated parent ion and product ions with one, two, and three molecules of 1,6-diaminohexaneattached, respectively.
+
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991 5H)5t
1975
(M+4H+B)4t
I
(M+4H)4t
L 00
1600
2000
2400
mh
mi2
(B+H)+
(b)
29891
10581
(b) P
= (Mt4H-17)
mh
mh Flguro 4. ES mass spectra of bovlne insulin acquired (a) in the absence of base and (b) in the presence of base with a total reaction tlme of 0.5 s.
+
An MS3 experiment with the (M 4 H)4+ parent ion involving CID followed by an ion/molecule reaction period illustrates the use of a clustering reaction to determine the charge state. By far the most intense product ion formed from CID of (M + 4 HI4+ (mlz 1434.5) falls at m/z 1430. This product can be formed from the parent with over 50% efficiency. Figure 5b shows the MS3 spectrum acquired after the parent ion was subjected to collisional activation and the product ion at m / z 1430 was mass-selected and allowed to react for 0.3 8. This spectrum, like the MS/MS spectrum of the parent ion, shows a series of peaks comprising, in this case, the unsolvated product ion and second generation product ions with one, two, and three molecules of 1,Gdiaminohexane attached, respectively. The charge, n,of the ions that make up the series of the peaks can be determined by dividing the mass of the base, mB, by the absolute value of the difference in m/z values for any two adjacent peaks, Le., where m/zl and m/zz indicate the m/z values for any two adjacent peaks. In both cases depicted in Figure 5, the ion charge is +4. This was already known, of course, from the mass spectrum for the parent ion but was not known for the product ion in the CID MS/MS spectrum. A charge of +4 for this ion indicates that the mass of the ion is 5720 f 4 Da. Either water loss or ammonia loss can account for this fragmentation. Experience with MS/MS of small peptides, where mass accuracy is sufficient to distinguish between water loss and ammonia loss, would suggest that this fragmentation is due to ammonia loss. An MS3 experiment with the (M + 4 H)4+ parent involving two stages of CID shows that the 5720-Da product ion fragments efficiently to a second generation product at m/z 1426. An MS4 experiment that allows the second generation product ion to react with the base indicates that it also is quadruply charged. Its mass is therefore 5703 f 4 Da, indicating either a loss of water or a loss of ammonia from the first generation product ion.
+
Flguo 5. Comparison of (a)the MS/MS spectnwn of the (M 4 H)'+ ion of bovine insulin whereby the parent ion is allowed to react wlth base and (b) the MS' spectrum whereby the mass-selected (M 4- 4 H)'+ lon undergoes C I D and the m / z 1430 product ion is mass-seiected and allowed to undergo reactbn with the base.
These examples show how a clustering reaction can be used to determine product ion charge states with only a minor loss in sensitivity. The CID steps are each greater than 50% efficient and the ion/molecule reaction experiments reault in essentially no ion loss. A minor decrease in sensitivity also results from adding a step for the ion/molecule reactions due to a lower duty cycle (see the discussion of the myoglobin results for more about the effect of duty cycle on sensitivity). Unfortunately, the (M + 4 HI4+ion does not fragment efficiently to structurally informative products. Other product ions are formed by CID of the (M+ 4 H)'+ ion and ita major product and second generation product, but they are all of less than a few percent intensity. CID of the (M 5 HY+ ion also gives an abundant loss of water or ammonia, but in contrast to the CID of the (M + 4 H)4+ion, it gives a richer variety of other producta and in greater abundance (23). A series of MS3experiments involving CID followed by ion/molecule reactions with 1,6-diaminohexane with a limited number of product ions show that the product ions tend to behave qualitatively s i m i i to the parent ions in their respective charge states. That is, +5 product ions tend to transfer a proton to the base and show a small tendency toward clustering. The quadruply charged producta tend to cluster. Product ions deemed to hold three or fewer charges, based on a comparison of the observed product ion m/z values with the expected A, B, C, X, Y,Z products from bovine insulin, tend not to react. These observations are consistent with the melittin data and our other data with ion/molecule reactions in that the likelihood for proton transfer tends to increase with charge state, as expeded based on destabilization of protons in the Coulombic field. Clustering tends to occur when the rate constant for proton transfer becomes small (
